In the clinical treatment and diagnosis of disease, assay systems which detect and quantitatively measure biologically important molecules are frequently employed, e.g., immunoassays and DNA/RNA detection assays. Liposomes provide certain advantages when used in these assay systems. For example, the use of liposomes can increase assay sensitivity because of the large number of marker molecules which can be encapsulated within a liposome vesicle.
Several types of marker molecules have been encapsulated in liposome vesicles including enzymatic, colorimetric, chromogenic, fluoregenic, radiometric, bioluminescent and chemiluminescent marker molecules. Chemiluminescent marker molecules, such as acridinium esters, have increasingly become the marker molecules of choice in clinical assay systems because they impart high sensitivity and wide linear range to most assays.
The marker molecules encapsulated within the liposome vesicle are most often released from the liposomes by complement-mediated vesicle lysis. See, e.g., U.S. Pat. No. 4,342,826. Other known methods for releasing the marker molecules from the liposomes include the use of lipolytic enzymes to lyse the vesicles. However, these known procedures are frequently inadequate for certain types of assays. For example, the use of liposomes with encapsulated chemiluminescent markers in a solid phase immunoassay requires a quicker and more complete release of the marker molecule than is provided by the above-identified methods.
U.S. Pat. No. 4,372,745 describes the use of liposomes with encapsulated fluorescent compounds in an immunoassay. This assay describes the use of a detergent (TRITON X-100) to break the liposome vesicles and release the fluorescent compounds. (TRITON is a registered trademark of the Rohm and Haas Company. TRITON X-100 is a non-ionic detergent of the polyethylene glycol p-isooctylphenyl there type having the formula EQU (CH.sub.3).sub.3 CCH.sub.2 C(CH.sub.3).sub.2 (C.sub.6 H.sub.4)O(CH.sub.2 CH.sub.2 O).sub.x H
where x averages 10.) See also U.S. Pat. Nos. 4,372,745 and 4,707,453 (use of TRITON X-100 to rupture liposomes), and U.S. Pat. No. 4,704,355 (use of saponin detergent to rupture liposomes). This procedure is not effective for use with certain marker molecules, such as chemiluminescent compounds, because the detergent (and certain other organic lytic agents like dimethyl sulfoxide) generate a high background signal (noise).
U.S. Pat. No. 4,927,769 (Application Ser. No. 071,660, filed on July 8, 1987), describes a method for enhancing the chemiluminescent signal of an acridinium ester which comprises oxidizing the acridinium ester in the presence of an effective amount of an enhancer selected from the group consisting of a cationic surfactant, a nonionic surfactant, and a sulfated primary alcohol.
Accordingly, it is an object of this invention to provide a novel method for releasing a marker molecule from within a liposome vesicle, which is quicker, more complete and generates less background interference than known methods for such release.
It is an another object of the present invention to provide a novel method for enhancing the chemiluminescence of acridinium esters using water-miscible alcohols.